scholarly journals Extraction, Characterization and Partial Purification of L-Asparaginase from the Leaves of Arachis Hypogaea L.

2019 ◽  
Vol 16 (3) ◽  
pp. 681-691
Author(s):  
Niranjana. J Niranjana ◽  
Kandasamy Arun Gandhi ◽  
D. Sunmathi ◽  
P. Nanthavanan
Keyword(s):  
Metal Ions ◽  
Arachis Hypogaea ◽  
Specific Activity ◽  
Lymphoblastic Leukemia ◽  
Gel Filtration ◽  
Size Exclusion ◽  
Partial Purification ◽  
Crude Enzyme Extract ◽  
Ammonium Sulphate Precipitation ◽  
Arachis Hypogaea L

L-asparaginase has been a promising therapeutic agent in the treatment of acute lymphoblastic leukemia and lymphoma. In recent times, due to the side effects of commercially available bacterial L-asparaginase and its unavoidable importance, plants are being explored as the source of L-asparaginase. The enzyme L-asparaginase was partially purified from Arachis hypogaea L. The crude enzyme extract was subjected to different purification steps including ammonium sulphate precipitation, dialysis followed by separation on Sephadex G-100 gel filtration (size exclusion chromatography) to obtain partially pure form of L - asparaginase. The enzyme was partially purified to 118 folds and contained specific activity of 4686.86 U/mg with 9.85% yield. SDS-PAGE electrophoresis of the partially purified enzyme revealed that it was a single protein with molecular weight of 70 kDa. The study on physiochemical properties showed that L - asparaginase from Arachis hypogaea L. was potassium-dependent in nature, where its optimum pH of enzyme activity was found to be 8.0 and temperature as 40°/50°C with reaction time of 15 - 20 minutes. Also it was observed that the L-asparaginase activity increased with the presence of metal ions such as Na+, Mg++, making it an enzyme dependent on metal ions for its reaction. In addition to this, it was revealed that the enzyme was partially inhibited in presence of certain chelators. The specificity of L-asparaginase obtained from Arachis hypogaea L. with lack of urease activity and minimal glutaminase activity along with less cytotoxicity on human blood indicated it as an efficient chemotherapeutic agent that could be investigated further in future studies.

scholarly journals New Lipase for Biodiesel Production: Partial Purification and Characterization of LipSB 25-4

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Aysel Ugur ◽  
Nurdan Sarac ◽  
Rukiye Boran ◽  
Berk Ayaz ◽  
Ozgur Ceylan ◽  
...  
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Nitrogen Sources ◽  
Biodiesel Production ◽  
Partial Purification ◽  
Period Effect ◽  
Carbon And Nitrogen ◽  
Ammonium Sulphate Precipitation ◽  
Thermophilic Conditions

The lipolytic activities of 300 Streptomyces isolates were determined in Tributyrin and Rhodamine-B Agar. Lipase activities were also measured with p-nitrophenyl palmitate (p-NPP) as a substrate. The strain of Streptomyces bambergiensis OC 25-4 used in this study was selected among 300 strains of Streptomyces from MUCC as the best lipase producer. The incubation conditions were optimized and the inoculum amount, incubation period, effect of carbon and nitrogen sources, and rates of MgSO4 and CaCO3 were investigated. LipSB 25-4 (the lipase produced by S. bambergiensis OC 25-4 strain) was partially purified with ammonium sulphate precipitation, dialysis, and gel filtration chromatography 2.73-fold and with 92.12 U/mg specific activity. The optimal pH and temperature for LipSB 25-4 were determined as 8.0 and 50°C, respectively. The lipase has high stability in all pH and temperature values used in this study. While LipSB 25-4 was slightly activated in the presence of β-mercaptoethanol, it was slightly reduced by PMSF. The enzyme conserved approximately 75% of its activity at the end of 60 h, in the presence of methanol and ethanol. Since LipSB 25-4 displays high activity in the thermophilic conditions and stability in the presence of organic solvents, this lipase can catalyse the biodiesel production from olive oil by the transesterification reactions.

Partial purification and properties of apoplastic β-1,3 glucanases of groundnut leaves treated with glucan isolated from a biocontrol agent, Acremonium obclavatum

2000 ◽  
Vol 78 (2) ◽  
pp. 168-174 ◽  
Author(s):  
M Sathiyabama ◽  
R Balasubramanian
Keyword(s):  
Arachis Hypogaea ◽  
Partial Purification ◽  
Ph Optimum ◽  
Native Page ◽  
Puccinia Arachidis ◽  
Ammonium Sulphate Precipitation ◽  
Arachis Hypogaea L ◽  
Purification And Properties ◽  
Molecular Masses ◽  
Electrophoretic Techniques

Apoplastic β-1,3 glucanases (G1, G2) of Arachis hypogaea L. (peanut) leaves treated with glucan have been partially purified by ammonium sulphate precipitation, sephadex G-100, CM-Sephadex, DEAE-Sephadex chromatography, and preparative native PAGE electrophoretic techniques. The pI values of purified enzymes G1 and G2 were near 8 and 4, respectively. The apparent molecular masses of purified glucanases G1 and G2 from glucan-treated peanut leaves were 36 and 34 kDa, respectively. Both isoforms (G1 and G2) showed their pH optimum of 5.0 and temperature optimum of 40°C. The partially purified enzymes hydrolysed laminarin better than other substrates and inhibited uredospore germination of Puccinia arachidis. Both isoforms (G1 and G2) inhibited spore germination of some biocontrol agents such as Acremonium obclavatum W. Gams, Myrothecium verrucaria (Alb. Schw.) Ditmer, Fusarium solani (Mart.) Sacc., and Trichoderma harzianum Rifai.Key words: Acremonium obclavatum, Arachis hypogaea, β-1,3 glucanase, glucan, inhibition, Puccinia arachidis.

scholarly journals Purification and characterization of fat body lipase from the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae)

2019 ◽  
Vol 81 (1) ◽  
Author(s):  
Rahma R. Z. Mahdy ◽  
Shaimaa A. Mo’men ◽  
Marah M. Abd El-Bar ◽  
Emad M. S. Barakat
Keyword(s):  
Metal Ions ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Gel Filtration ◽  
Molecular Weights

Abstract Background Insect lipid mobilization and transport are currently under research, especially lipases and lipophorin because of their roles in the production of energy and lipid transport at a flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from the last larval instar of Galleria mellonella. Results Purification methods by combination of ammonium sulfate [(NH4)2SO4] precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633 ± 0.000551 mg/ml and 1.5754 ± 0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in the purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore, biochemical characterization of fat body lipase was carried out through testing its activities against several factors, such as different temperatures, pH ranges, metal ions, and inhibitors ending by determination of their kinetic parameters with the use of p-nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35–37 °C and 37–40 °C and pH ranges of 7–9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+, and Na+ metal ions indicating that fat body lipase is metalloproteinase. Lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfonyl fluoride (PMSF), ethylene-diaminetetractic acid (EDTA), and ethylene glycoltetraacetic acid (EGTA) providing evidence of the presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 0.316 Umg− 1 Vmax and 301.95 mM Km. Conclusion Considering the purification of fat body lipase from larvae and the usage of some inhibitors especially ion chelating agents, it is suggested to develop a successful control of Galleria mellonella in near future by using lipase inhibitors.

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

scholarly journals Cardioactive Peptides from the CNS of a Caterpillar, the Tobacco Hornworm, Manduca Sexta

1985 ◽  
Vol 114 (1) ◽  
pp. 397-414
Author(s):  
Nicholas Platt ◽  
Stuart E. Reynolds
Keyword(s):  
Manduca Sexta ◽  
Specific Activity ◽  
Gel Filtration ◽  
Corpora Allata ◽  
Performic Acid ◽  
Partial Purification ◽  
Relative Molecular Mass ◽  
Two Factors ◽  
Frontal Ganglion ◽  
Manduca Sexta Larvae

1. A semi-isolated caterpillar heart bioassay was used to detect the presence of endogenous cardioactive material in the CNS of Manduca sexta larvae. 2. Cardioactivity was detected in all nervous tissue examined. Most activity (about 70% of the total in the CNS) was in the ganglia of the abdominal nerve cord (ANC). Cardioactivity was also detected in the abdominal transverse nerves, the proctodeal nerves and the corpora cardiaca/corpora allata. The source with the highest specific activity was the frontal ganglion. 3. Two factors, separable by Sephadex gel filtration, were distinguished in extracts of ANC: CAF 1, which has an estimated relative molecular mass (Mr) of about 4000, and CAF2 for which Mr is probably less than 1000. Both factors are apparently peptides. Neither is similar to any known insect cardioaccelerator. 4. Both CAF 1 and CAF 2 are able to cause cardioacceleration when injected into tetrodotoxin-paralysed caterpillars. 5. CAF 2 is present in both larvae and in adults. CAF 1 is present only in the caterpillar. The larval heart responds to both factors; the adult heart responds only to CAF 2. 6. Partial purification of CAF 1 and CAF 2 by reverse-phase HPLC gives a single peak of bioactivity in each case. 7. The biological activity of CAF 1 is destroyed by α-chymotrypsin, but not by trypsin. CAF 2 is not attacked by trypsin or by α-chymotrypsin. Treatment with performic acid or cyanogen bromide destroys the activity of both CAF 1 and CAF 2.

scholarly journals Optimization of Metal Ions in Sugarcane Bagasse Fermenting Medium for the Production of Streptokinase by Streptococcus equisimilis

2021 ◽  
Vol 9 (2) ◽  
pp. 24-30
Keyword(s):  
Metal Ions ◽  
Sugarcane Bagasse ◽  
Specific Activity ◽  
Gel Filtration ◽  
Value Added ◽  
Cost Effective ◽  
Fermentation Medium ◽  
Exchange Chromatography ◽  
Sds Page ◽  
The Cost

Streptokinase is a fibrinolytic enzyme and a product of β-hemolytic Streptococci strains. This enzyme is used as a medication to break down clots in some cases of heart disease. Streptococcus equisimilis, a species of group C Streptococci, is widely used for the production of streptokinase by fermentation technology. In this study, the sugarcane bagasse fermentation medium was optimized for metal ions (KH2PO4, MgSO4.7H2O, CaCO3 and NaHCO3) at various levels to attain the maximal production of streptokinase. Sugarcane bagasse was used due to its profuse availability and as an ideal substrate for microbial processes for the manufacturing of value-added products. The results showed that maximal streptokinase production was found at 0.04% KH2PO4, 0.04% MgSO4.7H2O, 0.15% NaHCO3 and 0.04% CaCO3. Finally, the optimized medium resulted in 84.75 U/mg specific activity and 74.5% recovery. The purification process was carried out simultaneously using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. Finally, a purified sample of streptokinase was run on SDS-PAGE and resolute 47 kDa molecular weight. The use of β-hemolytic Streptococci to obtain streptokinase is not free from health risks and is related to anaphylaxis. This study provides a way forward for the cost-effective ways to obtain streptokinase for the treatment of thrombosis.

Partial Purification and Some Properties of a Hydroxycinnamoyl Glucosyltransferase from Tomato Fruits

1980 ◽  
Vol 35 (11-12) ◽  
pp. 967-972 ◽  
Author(s):  
A. Fleuriet ◽  
J. J. Macheix ◽  
R. Suen ◽  
R. K. Ibrahim
Keyword(s):  
Ferulic Acid ◽  
Metal Ions ◽  
Deae Cellulose ◽  
Divalent Metal ◽  
Partial Purification ◽  
Divalent Metal Ions ◽  
Tomato Fruits ◽  
Ammonium Sulphate Precipitation ◽  
Sh Group ◽  
Cherry Tomatoes

A glucosyltransferase was isolated from immature “cherry” tomatoes and was partially purified (200-fold) by ammonium sulphate precipitation and successive chromatography on Sephadex G-100 and DEAE-cellulose columns. The enzyme utilised the free hydroxycinnamic acids and UDP-glucose in the formation of their respective glucosides (pH 8.0) and glucose esters (pH 7.0); but did not accept the CoA thiolesters of HCAs in the presence of glucose-1-phosphate. The constant glucoside/glucose ester ratio observed during purification suggests that both reactions are catalysed by the same enzyme. The Km values for ρ-coumaric, caffeic, ferulic and sinapic acids were 0.8, 1.5, 1.4 and 2.5 μᴍ, respectively. With ferulic acid as substrate, the Km value for UDPG was 10 μᴍ. The enzyme required an -SH group for activity and the reaction was strongly inhibited by EDTA, divalent metal ions and UDP.

scholarly journals Partial purification of topoisomerase IB from serum of diabetic patients and study it's kinetic properties and molecular weight

2019 ◽  
Vol 23 (10) ◽  
pp. 46
Author(s):  
Saif M. Hasan ◽  
Firas T. Maher ◽  
Nagham Q. Kadhim
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Maximum Velocity ◽  
Partial Purification ◽  
Diabetic Patients ◽  
Kinetic Properties ◽  
Topoisomerase Ib ◽  
Electrophoresis Method

This study was done to partially purification of  topoisomerase IB from serum of diabetic patients using Gel filtration technique, by using Sephadex G 100 gel. A single peak in fraction four has been obtained, and the degree of purification (17.1) fold, enzyme yield (108.2%) and specific activity (0.189ng/mg). Kinetics studies for the partial purified enzyme were carried out which showed optimal concentration of  substrate which was (0.1ng/ml), Michael's - Menten constant (Km=0.033ng) and maximum velocity (Vmax=0.90 ng/ml), while optimum Temperature was (37C°) and optimum pH was (7.5). The molecular weight of the partial purified enzyme has been determined by gel electrophoresis method, in presence of polyacrylamide  gel and sodium dodecyl sulphate (SDS-PAGE) which showed that the approximated molecular weight was (66KD).   http://dx.doi.org/10.25130/tjps.23.2018.168 

scholarly journals Leucine Aminopeptidase from Arachis hypogaea L. Seeds Partial Purification and Characterization

2017 ◽  
Vol 33 (5) ◽  
pp. 2676-2680 ◽  
Author(s):  
Taghreed U. Mohammd ◽  
Layla O. Farhan ◽  
Ashgan S. Dawood ◽  
Bushra F. Hasan
Keyword(s):  
Arachis Hypogaea ◽  
Leucine Aminopeptidase ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Arachis Hypogaea L

scholarly journals Purification and characterization of fat body lipase from the Greater Wax Moth Galleria mellonella (Lepidoptera: Pyralidae)

2019 ◽  
Author(s):  
Rahma R.Z. Mahdy ◽  
Shaimaa A. Mo’men ◽  
Marah M. Abd El-Bar ◽  
Emad M.S. Barakat
Keyword(s):  
Metal Ions ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Gel Filtration ◽  
Molecular Weights

AbstractLipid mobilization and transport in insects is under investigation, especially lipases and lipophorin because of their roles in energy production and transport of lipids at flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from last larval instar of Galleria mellonella. Purification methods by combination of ammonium sulfate precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633±0.000551 mg/ml and 1.5754±0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore biochemical characterization of fat body lipase was carried out through testing its activities against several factors such as; different temperatures, pH ranges, metal ions and inhibitors ending by determination of their kinetic parameters with the use of p-Nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35-37°C and 37-40°C and pH ranges of 7-9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+ and Na+ metal ions indicating that fat body lipase is metalloproteinase. Additionally, lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfony fluoride (PMSF), ethylene-diaminetetractic acid (EDTA) and ethylene glycoltetraacetic acid (EGTA) providing an evidence of presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 301.95mM Km and 0.316 Umg−1 Vmax. By considering the purification of fat body lipase from larvae and using some inhibitors especially ion chelating agents, it is suggested to develop this study by using lipase inhibitors to reach a successful control of Galleria mellonella in the near future.

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